Detection of equine herpesvirus antibodies in large‐scale donkey farms in Liaocheng area

Abstract Background Equine herpesvirus (EHV) can cause respiratory, reproductive and neurological diseases in equine animals, including donkeys. The main pathogens responsible for these diseases are EHV type 1 (EHV‐1) and EHV‐4. In this study, we collected serum samples from 230 donkeys on 27 large‐scale donkey farms to detect EHV‐1 and EHV‐4 antibodies. We analyzed the presence of EHV antibodies based on region, age and season. Results Out of the 27 farms, 62.96% (17/27) tested positive for EHV. Of the 230 donkeys tested, 2.61% (6/230) were positive only for EHV‐1, 5.22% (12/230) were positive only for EHV‐4, and 4.78% (11/230) were positive for both EHV‐1 and EHV‐4. The highest percentage of positive donkeys (21.28%) was found in Dong'e County. The seropositivity rate among donkeys aged 1–4 years was significantly higher compared to the group of donkeys aged 0–1 year (p < 0.05). Additionally, the positive rate was significantly higher in fall and winter compared to spring and summer (p < 0.05). Conclusions Altogether, our findings indicate that large‐scale donkey farms in the Liaocheng area have a high prevalence of EHV antibodies. Since Liaocheng is an important donkey trading market in Shandong Province, it is crucial to consider the risk of disease transmission based on our test results. This will help in early detection and prevention of EHV outbreaks.

EHV was first identified and reported in 1933 in the United States Since then, the virus has been detected in several countries and regions (Studdert et al., 1986;Turan et al., 2012).In the United States, it has been reported that approximately 80%-90% of horses have been infected with either EHV-1 or EHV-4 (Allen et al., 2008).EHV-1 was found in Thoroughbred horses with respiratory disease in Hong Kong and was reported for the first time in China (Mason et al., 1989).In 2019, an outbreak of EHV-1 infection in the Ili Kazakhstan Autonomous Region in Xinjiang, China, resulted in EHV myeloencephalopathy (EHM) and abortion, affecting 45 horses (Hu et al., 2022).
Additionally, an abortion "storm" (43 out of 800) in Yili horses in Zhaosu County, northern Xinjiang, China, was ultimately confirmed to be caused by EHV-1 infection in 2021 (Tong et al., 2022).Donkeys, as notable members of the Equidae family, have also been reported to be infected with EHV-1 and EHV-4 (Harless & Pusterla, 2006).Consequently, Ali et al. (2020) isolated EHV-1 from the foetus of an aborted female donkey in Egypt.Furthermore, Yildirim et al. (2015) conducted EHV investigations on donkeys in the northeastern region of Turkey and found high positivity rates for EHV-1 and EHV-4.
Although reports of EHV infection in donkeys are limited, EHV poses a significant threat to donkey health.The donkey breeding industry has recently experienced rapid development in Shandong Province, with large-scale farming of donkeys in Liaocheng city leading the way.
However, the transition from traditional breeding to large-scale production is hindered by the issues such as abortion of pregnant female donkeys, diarrhoea in donkey foals and rhinopneumonia infection in donkeys and horses.To date, there has been no sero-epidemiological survey of EHV in large-scale donkey farms in China.Therefore, this study aims to determine the degree of EHV antibody positivity in largescale donkey farms in Liaocheng city.The study will also analyze the risk factors affecting EHV infection in order to provide reference data for the prevention and control of EHV. and cities, all located in the northern hemisphere with a temperate monsoon climate.From these donkeys, we randomly selected 230 individuals that had not received immunization against EHV.We obtained venous blood samples from these selected donkeys with the consent of their respective owners from 27 different donkey farms.Blood was collected using negative pressure blood collection tubes, and after clot-ting, the blood was centrifuged at 2000 rpm for 10 min to obtain serum.

Sample collection
The serum samples were then stored frozen at −80 • C for further use.

Detection of EHV-1/4 antibodies
An enzyme-linked immunosorbent assay (ELISA) was employed to detect EHV1/4 antibodies in 230 serum samples utilising the EHV1/EHV4-Ab antibody assay kit (SVANOVIR EHV1/EHV4-Ab; Svanova AB).The procedure was performed according to the manufacturer's instructions provided with the kit.The results were determined by measuring the optical density of the serum samples at 450 nm.

Data analysis
Based on the ELISA results, we investigated antibodies against EHV1 and EHV-4 in terms of region, age, and season.SPSS 17.0 was used for the statistical analysis of the data, and 95% confidence interval were calculated.The chi-square test (χ2) was used to assess significant differences in EHV-1/4 prevalence between different regions, ages and seasons.Note that p-values <0.05 are considered significant.Finally, we utilized GraphPad Prism 8 software to construct the figures.

Association with risk factors
Based on the simplified official map of Liaocheng area, an EHV positivity map was created using the positivity rate from each region as detailed in Table 1 (Figure 1a).The map shows that the highest EHV antibody positivity rate of 21.28% (10/47) was observed in large-scale donkey farms in Dong'e County.In other regions, the EHV antibody positivity ranged from 8.33% to 13.95%.Although horses are the main hosts of EHV-1 and EHV-4, donkeys may be infected with EHV through contact with horses, resulting in antibody positivity (Azab et al., 2019;Cano et al., 2008;Diallo et al., 2006Diallo et al., , 2007)).
The rate of donkey EHV seropositivity varied in different countries and regions (Getachew et al., 2014;Lazić et al., 2023;Mekonnen et al., 2017;Wegdan et al., 2016).Our analysis revealed that Dong'e County had the highest number of positive cases of EHV, which might be attributed to the high density of donkeys in the area.Dong'e County accounts for over 50% of the total donkey population in Liaocheng.
These results suggest that areas with a high density of donkeys exhibit a higher seropositivity rate, indicating a need for increased attention in these regions.Foote et al., 2003) EHV antibody positivity was age-dependent on large-scale donkey farms in Liaocheng, with donkeys aged 1-4 years being more likely to test positive.In the present study, donkeys of all ages were at risk of seropositivity, with the highest seropositivity in adult donkeys aged 1-4 years, probably related to the disappearance of maternally derived antibodies.In addition, we found that on large-scale donkey farms, the total number of positive serum tests collected during the fall and winter seasons was significantly higher than the total number of positive rates observed during the spring and summer seasons.Suggesting a seasonal pattern in EHV distribution among donkey herds.This seasonal variation may be related to the temperate monsoon climate of Liaocheng.During the fall and winter seasons, the decrease in temperature causes stress in donkeys, which reduces their resistance to external factors (Chenchev et al., 2011).Consistently, ies in surrounding animals (Costantini et al., 2018;Ruiz-Saenz et al., 2008).

CONCLUSION
In

F
The equine herpesvirus (EHV) antibodies test results.(a) Schematic diagram of EHV antibodies positive rate in Liaocheng area.(b) Pie charts of EHV antibody positivity rate of different age groups in Liaocheng city.(c) Line graph of EHV antibody positivity rate in Liaocheng in different seasons.TA B L E 2 p-values and χ 2 for equine herpesvirus (EHV) positivity between different regions.

Foote
et al. (2003) revealed that external factors further spread the EHV infection during transportation and under stress, resulting in an increased rate of animal seropositivity.Liaocheng donkey farms primarily import donkeys from the Balinzuo Banner and Arukorqin Banner of Chifeng city in Inner Mongolia, as well as Jianping County of Fuxin city in Liaoning Province.These animals are transported by land to various donkey farms in Liaocheng.Stress caused by longdistance transport and temperature changes can easily lead to EHV infection and transmission, resulting in the production of antibod- conclusion, the results of our survey indicate that antibodies against EHV-1 and EHV-4 are prevalent in large-scale donkey farms in Liaocheng.Our study also analyzed various factors that could potentially affect this prevalence.These findings are crucial for guiding the development of strategies to prevent and control EHV.The high rate of EHV antibody positivity in donkey serum warrants considerable attention.It is vital for these farms to adopt measures to eliminate possible transmission routes.Additionally, strict practices such as isolation, observation, and quarantine should be enforced when introducing donkeys.AUTHOR CONTRIBUTIONS Yanfei Ji: Conceptualization; data curation; formal analysis; visualization; writing-original draft; investigation; software.Xia Zhao: Conceptualization; methodology; supervision; writing-review and editing; validation; resources.Wenqiang Liu: Methodology; investigation; supervision; project administration; writing-review and editing; funding acquisition.
Statistics of positive rates of EHV-1 and EHV-4 (where EHV is equine herpesvirus) antibodies in Liaocheng city, China.
(Kubacki et al., 2021)anying pie chart (Figure1b) show that donkeys aged 1-4 years had the highest antibody positivity rate, significantly higher than other age groups, with a notable difference observed in the 0−1-year age group (p < 0.05).The line graph in Figure1cdepicts the seasonal serum testing results over four consecutive seasons from 2019 to 2020.The data demonstrate higher positivity rates in fall and winter compared to spring and summer.The chi-square and p-values for the comparison of different regions, seasons and age groups are shown in Tables2-3.TA B L E 1the transmission of infectious diseases in the large-scale donkey farms located in Liaocheng city.By doing so, we can effectively safeguard the economic stability of the donkey industry in Shandong Province.In 2021, an outbreak of EHV-1 was reported in Spain, resulting in infections and fatalities in horses across more than 10 countries(Kubacki et al., 2021).Despite these significant outbreaks, there is a notable lack of investigation into EHV serostatus in donkeys in China.The preva-lence and impact of EHV infection in donkeys remain unknown.In this study, 230 serum samples were tested, revealing that exposure to EHV infection is common in large-scale donkey farms in the Liaocheng area.
Summary of seroprevalence of equine herpesvirus (EHV) (EHV1 and EHV4) in donkeys in Liaocheng, China, and other countries.